1. Field of the Invention
The invention relates to a method of detecting the formation of inclusion bodies such as sequestosomes. The invention also relates to a method of detecting inclusion bodies in neurodegenerative diseases. The invention further relates to a method of screening for therapeutic agents that disperse the inclusions.
The invention relates to transgenic non-human animals and transgenic non-human animal cells harboring a transgene containing a mutation in the p62 gene and having a functionally disrupted p62 gene locus. The invention further relates to transgenes and targeting constructs used to produce such transgenic animals and cells, methods of using such animals for modeling aging related disorders, and methods for using such animals to produce transgenic nonhuman animals and cells including at least one further transgene. The invention further relates to functions of p62 first revealed by phenotypes generated in such transgenic animals and cells such as in development of obesity, type 2 diabetes mellitus, non-alcoholic fatty liver, various tumors, increased male mortality, intracellular inclusion named sequestosome, and redox regulation.
The invention further relates to methods of diagnosing early stage of, predisposition to or susceptibility of a mammalian subject to an aging related disorder or disease.
2. General Background and State of the Art
Cloning and sequencing of human p62 gene is described in U.S. Pat. Nos. 6,291,645B1 and 5,962,224, which are incorporated by reference herein in their entirety. The gene product, protein, in human is p62 and the gene name is SQSTM localized at the human chromosome 5q35. The same gene products of mouse and rat are named differently as A170 and ZIP, respectively, and the gene encoding A170 is localized at the mouse chromosome 11, sqstm 1. For convenience, these genes and proteins of human and mouse will be named p62 gene and p62, respectively, in this manuscript.
Aging is associated with the degeneration of cells, tissues, and organs, resulting in diseases such as cancer, cardiovascular failure, obesity, type 2 diabetes mellitus, non-alcoholic fatty liver, and a number of neurodegenerative diseases, as well as the decline of most measures of physiological performance. It has become evident that the redox status of the cell is importantly involved in several basic cellular processes, such as signal transduction, gene expression and thereby cell proliferation and apoptotic cell death. The accumulation of somatic damage is considered a main cause of the aging process. Among the various sources of somatic damage, reactive oxygen species (ROS), the natural by-products of oxidative energy metabolism, are often considered as the ultimate cause of aging.
Obesity causes many health problems. Obesity is the presence of excessive amount of adipose tissue and has become a significant human health problem in the modern world. Excessive adipose tissue causes, both independently and in association with the development of type 2 diabetes mellitus, coronary heart disease (CHD), an increased incidence of certain forms of cancer, respiratory complications and osteoarthritis. Obesity can result from a derangement in one or more of the three components of energy balance: energy intake, energy expenditure, and energy partitioning. Coordinated regulation among these three components is achieved through neuronal network and neuro-endocrine system, and any defect in these components is sufficient to cause obesity.
Diabetes is defined as a state in which carbohydrate and lipid metabolism are improperly regulated by insulin. This results in elevated fasting and postprandial serum glucose that leads to complications if left untreated. There are two major categories of the disease, Types 1 and 2. Type 2 diabetes is far more common and results from a combination of defects in insulin secretion and action. Type 2 diabetes is characterized by a progressive decrease in insulin action, followed by an inability of the β cell to compensate for insulin resistance. Insulin resistance is the first lesion, due to interactions among genes, aging, and metabolic changes produced by obesity. Insulin resistance in visceral fat leads to increased fatty acid production, which exacerbates insulin resistance in liver and muscle. The β cell compensates for insulin resistance by secreting more insulin. Ultimately, the β cell can no longer compensate, leading to impaired glucose tolerance, and diabetes.
A majority of individuals suffering from type 2 diabetes are obese, with central visceral adiposity, and an imbalance in energy intake and expenditure that leads to numerous metabolic abnormalities. Insulin resistance might be the result of obesity, but might also contribute to its development. Recent insights into the biology of the adipocyte as an endocrine organ have supported this latter idea. It is now known that this cell type is more than a storage site for lipids; it also secretes a number of important circulating factors, including leptin, TNFα, angiotensin, and PAI-1, and is the major source for endogenous production of nonesterified fatty acids (NEFA) via lipolysis. Indeed, the dynamic interactions between the adipocyte, certain nuclei of the ventromedial hypothalamus and the organs of insulin synthesis and action insure the coordinated regulation of insulin sensitivity, as well as energy intake and expenditure.
Non-alcoholic fatty liver disease (NAFLD) encompasses two histological lesions: fatty liver and steatohepatitis. Insulin resistance is suggested to be a key pathophysiological abnormality in patients with NAFLD. Insulin resistance results from a complex interplay between the major targets of insulin action, i.e. muscle, adipose tissue and liver, versus the ability of the pancreatic islet beta cells. The metabolic and clinical profile associated with insulin resistance is thus defined by the factors that produce and maintain insulin resistance and the effects of decreased insulin sensitivity on various insulin-dependent pathways. The major metabolic defects associated with insulin resistance are increased peripheral lipolysis, increased hepatic glucose output due to increased gluconeogenesis and increased lipid oxidation. This is associated with an oxidative stress in the liver that may be compounded by additional pathophysiological abnormalities. Increased fatty acid beta oxidation as well as peroxisomal fatty acid oxidation can both lead to increased reactive oxygen species generation and subsequent lipid peroxidation. The oxidative stress produced by increased fatty acid oxidation may also produce additional harmful effects that amplify the disease process in the liver.
Increased oxidative stress and resulting changes in cellular redox status is also known to be an important factor in development of a number of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Intracellular inclusion (ubiquitin positive and/or negative) is an ultrastructural hallmark of such neurodegenerative disorders. The fact that proteasome immunoreactivity is augmented in most dystrophic neuritis in a variety of diseases, suggesting that ubiquitin-proteasome pathway is critically involved in this pathologic insoluble matter formation. In general, ubiquitinated or oxidized proteins do not accumulate in normall cells, and are rapidly degraded at the proteasome. Ubiquitinated or oxidized protein inclusions in the cell must result from a malfunction or overload of the proteasome pathway or from structural changes in the protein substrates, halting their degradation. However, the questions of what determines the fate of a protein in relation to the formation of such abnormal inclusion bodies and what would be the pathologic impact of inclusion bodies in neuro-degeneration process remain unclear. Thus, clues to these questions would lead to better understanding of neurodegenerative process and thereby would provide diagnostic and therapeutic tools.
Aging-related disorders, such as formation of intracellular inclusions, obesity, diabetes, cancer, in particular, liver cancer, fatty liver, Paget Disease of Bone, and early mortality for male continue to pose significant health problems. What is needed is a live animal model, which may be used for the study of these diseases and other age-related disorders for screening and evaluation of potential therapeutic agents as well as potential diagnostic/prognostic probes useful in the treatment of these disorders. These and other needs in the art are addressed by the present invention.